Characterization of a mouse serotonin 5-HT3 receptor purified from mammalian cells.
A serotonin 5-HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding approximately 50% of the histidine-tagged 5-HT3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild-type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of approximately 5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5-HT3 receptor is a pentameric homooligomer. The secondary structure of the 5-HT3 receptor as determined by circular dichroism appeared to consist of mainly alpha-helices (50%) and beta-strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.
Hovius
R
R
Chemistry Department, Swiss Federal Institute of Technology, Lausanne.
Tairi
A P
AP
Blasey
H
H
Bernard
A
A
Lundström
K
K
Vogel
H
H
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D014162:Transfection
D046911:Macromolecular Substances; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins